Project 4 has been a leader in revealing the myriad roles played by microRNAs in human cancer. We will build upon this foundation with an increased biological focus on breast cancer. We will isolate the six, well defined mammary epithelial cell types from normal virgin and parous female mice and define their miRNA and mRNA expression profiles. In so doing, we hope to better define the mammary stem cell (MaSC) and gain an appreciation for the pathways that maintain its self-renewal. This will include a definition of miRNAs and miRNA targets that are important in the MaSC. Using normal cells as a reference point, we will work to understand the relationship between mammary tumor initiating cells and normal mammary stem cells. In part, we aim to test the relevance of tumor initiating populations, which thus far have only been detected in transplantation studies, to breast cancer in in vivo models of basal tumorigenesis. We hope to identify pathways that determine tumor initiating potential and to relate these to self-renewal pathways used by mammary stem cells. We appreciate that both normal and tumor cells function in context, and we will therefore strive to understand how the in vivo niche supports MaSC self-renewal. Similarly, we will ask whether tumor initiating cells occupy a niche or whether their special status is a cell autonomous property. With the realization that it is usually metastatic disease that kills patients, we will also probe the niches occupied by disseminated tumor cells, which can lie dormant for decades following initial treatment before they progress to frank metastases. Using a series of highly innovative strategies, including molecular profiling, RNAi-based genetics, and sophisticated imaging a whole, this Project takes a comprehensive approach to understanding the roles of miRNAs in breast cancer.